| Intermedin (IMD), also called adrenomedullin2(ADM-2), is a novel memberof the calcitonin gene-related peptide (CGRP) superfamily. IMD is localized in thetissues of the cardiovascular and renal systems and exerts its biological effectsthrough the stimulation of complexes consisting of calcitonin receptor-like receptor/receptor activity-modifying protein complexes. This study is designed to explore thephysiological and pathological of IMD in rat renal tubular cellhypoxia-reoxygenation（H/R） injury. This study includes two parts:Part1Intermedin protects against tubular cell hypoxia-reoxygenationinjury through suppresses endoplasmic reticulum stress（ERS）-relatedapoptosis1H/R triggers ERS and induces cell apoptosis.Objective The hypoxia/reoxygenation（H/R） injury models of rat renaltubular epithelial cells（NRK-52E）were established to observe the expression of ERSmolecules and its receptors after IRI, and explore the effect of ERS in renal IRI.Methods The rat renal tubular epithelial cells （NRK-52E）were randomlydivided into control, H/R and tunicamycin（TM） groups. The H/R model inNRK-52E cells consisted of hypoxia for1h and reoxygenation for2h. The flowcytometry were employed to assess cell apoptosis. ER specific residentfluorochrome Dapoxyl was used to observe ER morphological changes.Realtime-PCR and Western blotting were used to detect expression of ERSmolecules such as GRP78, CHOP and Caspase12. Results⑴Compared with control, the apoptosis rate of H/R and TMincreased significantly（P<0.01.⑵Dapoxyl staining demonstrated that the structureof the ER was severely destroyed in H/R and TM, the fluorescence density showednonuniform distribution, accompanied by notable vacuoles in the ER.⑶Comparedwith control, the mRNA and protein expressions of GRP78, CHOP and Caspase12in H/R and TM groups were all up-regulated significantly（P<0.05）.Conclusion It was suggested that H/R as the same as Tm up-regulateexpression of GRP78, CHOP and Caspase12and induce cardiomyocyte apoptosis.And the strategy of decersed ERS may be beneficial for the renal IRI.2IMD suppresses ERS-related apoptosis induced by H/RObjective The H/R model in NRK-52E cells, which were transfected theeukaryotic expression vector of rat IMD, were observed the changes of ERS andinvestigate the molecule mechanism of IMD cytoprotection.Methods The rat renal tubular epithelial cells （NRK-52E）were randomlydivided into control, H/R, H/R+pIRES2and H/R+IMD groups. IMD wasover-expressed in NRK-52E cells using the vector pIRES2-EGFP/IMD.The flowcytometry were employed to assess cell apoptosis. ER specific residentfluorochrome Dapoxyl was used to observe ER morphological changes.Realtime-PCR and Western blotting were used to detect expression of ERSmolecules such as GRP78, CHOP and Caspase12.Results⑴I MD gene transfer markedly decreasedthe apoptosis indexcompared with that of H/R（P<0.05）.⑵Over-experss IMD decreased the injury ofthe ER.⑶Compared with H/R group, the mRNA and protein expressions ofGRP78, CHOP and Caspase12in IMD group were all down-regulated significantly（P<0.05）. Conclusion IMD suppressed the up-regulation of GRP78, CHOP and Caspase12and inhibited ERS-related apoptosis. It was suggested that suppression ofERS-related apoptosis was invlolved in cytoprotection of IMD.Part2Intermedin protects against tubular cell hypoxia-reoxygenationinjury in vitro by promoting cell proliferation and up-regulating cyclin D1expressionObjective To evaluate the effect of IMD on cell proliferation and regeneration ina cultured rat tubular epithelial cell line (NRK-52E) that was subjected tohypoxia-reoxygenation (H/R) injury.Methods The NRK-52E cells were divided into6groups. One of them wascontrol group; the other five model groups were exposed to H/R condition, followingthe intervention of single H/R, primitive vector, highly expressed IMD vector, ERKinhibitor（PD98059）, JNK and P38excitomotor（anisomycin）, respectively. Thecontent of LDH was detected to observe the influence of IMD on H/R injury and itsmolecule mechanism. The cell proliferation was detected by MTT. The cell cycle wasdetected by flow cytometry. Real-time PCR and Western blotting were used todetermine mRNA and protein levels. In addition, luciferase reporter assays andelectrophoretic mobility-shift assays were performed to measure cyclin D1promoteractivity and transcription factor activity.Results⑴LDH release assay was performed to measure the irreversible celldeath of NRK-52E cells. This assay also showed that IMD was protective against H/Rinjury. In comparison to the control, H/R treatment increased LDH activity by106%(P<0.01); in contrast, compared to H/R and H/R+pIRES2, IMD treatment decreasedLDH activities by34%and29%(P<0.01).⑵Cell viability was examined in differentgroups of H/R treatments. Compared to the H/R group, the over-expression of IMDmarkedly promoted cell survival and provided protection against H/R injury (80.22±4.42%vs.59.77±9.63%, P<0.05).⑶The Thr/Tyr phosphorylation states ofERK, JNK, and P38, corresponding to the activated forms of these enzymes, werefaintly detectable in control cells. In contrast, H/R increased the phosphorylatedproportions of ERK, JNK, and P38, whereas the over-expression of IMD reversedJNK and P38phosphorylation and caused ERK activation, which indicated that IMDcan regulate the balance of MAPK activities.⑷In NRK-52E cells following thedifferent treatments, the changes of detected index were shown:①The effect ofIMD in promoting cell proliferation and decreasing LDH content were weaken byERK inhibitor（PD98059）or JNK and P38excitomotor（anisomycin）.②Theproliferation of NRK-52E cells was significantly inhibited by H/R treatment. Incomparison to the control, H/R treatment of NRK-52E cells increased the proportionof cells in the G0/G1phase but decreased the proportion of cells in the S and G2/Mphases. Moreover, the over-expression of IMD resulted in S and G2/M phaseredistribution and the accumulation of G2/M-phase cells, and this modulation wassignificantly suppressed in the presence of PD98059, anisomycin. Together, theseresults indicate that IMD plays an important role in the regulation of cell-cycleprogression and cell division in NRK-52E cells experiencing.③The real-time PCRand Western blotting results indicated that the mRNAand protein expression levels ofcyclin D1and cyclin E were increased in H/R-treated cells. IMD further stimulatedthis up-regulated expression in NRK-52E cells. Furthermore, when cells were treatedwith the ERK inhibitor PD98059, or the JNK and P38agonist anisomycin, IMDfailed to maintain a stable expression of cyclin D1and cyclin E, indicating thatMAPK pathways are involved in the regulatory effects of IMD on cyclin D1andcyclin E expression.④NRK-52E cells transfected with a cyclin D1promoter-luciferase construct exhibited increased luciferase expression after H/Rtreatment. Compared to I/R alone, the over-expression of IMD further increased cyclin D1-luciferase activity, and this up-regulation of cyclin D1reporter activity inNRK-52E cells was abolished by the ERK inhibitor PD98059，the JNK and P38agonist anisomycin.⑤As measured by EMSA, IMD was shown to promote AP-1DNA-binding activity, and this modulation was significantly suppressed in thepresence of PD98059, anisomycin.⑥We also analyzed the presence and localizationof cyclin D1and found that this protein had a predominantly nuclear localization inNRK-52E cells, although cytoplasmic localization was also observed. Moreover, theover-expression of IMD increased the immunofluorescence staining of cyclin D1.Conclusion The study show that the over-expression of IMD may promoterenal cell proliferation and regeneration after renal tubular cell H/R injury via theup-regulation of cyclin D1, which may be mediated by the ERK, JNK, and P38signaling pathways. Thus, enhancing IMD, either by gene therapy or by infusion ofthe IMD protein, may provide a new therapeutic intervention strategy for renal H/Rand I/R injury. |
PDF Full Text Request